Project Summary/Abstract The immune system has developed to identify and target foreign or mutated intracellular proteins to defend against cancers, viruses and infections. Intracellular proteins are presented as peptides in major histocompatibility complexes on the surfaces of cells, which are recognized by T cell receptors (TCR) on CD8+ T cells. TCRs are attractive for cancer therapy because they can specifically target peptide-MHC (pMHC) antigens found across numerous patients and cancers, known as shared antigens. Shared antigens have been identified from cancer testis antigens and melanoma antigens. Endogenous TCRs targeting these antigens have been identified from patient samples. These TCRs have been successful as treatment in numerous patients when exogenously expressed in autologous T cells and reinfused. While, tumor reactive TCRs have a great potential for use in therapy, it remains challenging to identify the target of tumor reactive TCRs. This limits the repertoire of known shared antigens that can be targeted for therapy. Here we propose a high throughput method to identify the targets of TCRs, thereby increasing the potential of TCR based therapies and identifying novel shared antigens. To identify the targets of TCRs, a DNA library encoding peptides will be used to present a large array of representative pMHCs. The peptide libraries are designed to have variability in positions responsible for the majority of TCR contacts, biasing the libraries to be more reactive. To determine TCR targets in a functionally relevant manner, relying on the cytotoxicity of T cells, coculture depletion screens will be performed. Peptides depleted from the screens are considered hits. The hits will be used to identify peptide binding motifs related to TCR reactivity. From these motifs clinically relevant, potential targets will be identified. The targets will be validated using single peptide coculture killing assays and ELISpot for identification of interferon gamma, a marker of T cell reactivity. This technology will be used to identify clinically relevant targets of TCRs from tumor infiltrating lymphocytes.